Participation of cytochrome b5 in the liver microsomal elongation of fatty acids in the rat is suggested by: (a) an increase in the rate of reoxidation of liver microsomal cytochrome b5 following reduction by NADH, in the presence of both malonyl coenzyme A and ATP, and (b) a 60% inhibition of incorporation of malonyl-CoA into microsomal fatty acids in the presence of anti-cytochrome b5 IgG; control IgG was without effect. Consistent with this conclusion, there was no increase in oxygen consumption following the addition of malonyl CoA and ATP to liver microsomes, in contrast to the addition of stearyl-CoA which produced a 3-fold increase. Futhermore, anaerobiosis did not inhibit the malonyl-Co-a-stimulated reoxidation rate of b5, whereas it did inhibit by 95% the stearyl-CoA-stimulated reoxidation rate. A 2- to 4-fold increase in the rate of reoxidation of b5 by malonyl-CoA was observed in liver microsomes from animals maintained on a fat-free diet, with maximal activity occurring after 48 hours. This stimulation was not observed in the absence of ATP or when malonyl-CoA was replaced by malonic acid. The malonyl-CoA effect was linear with microsomal protein up to at least 2 mg/ml, and the km for the malonyl-CoA was approximately 2.0 microns M. Cyanide was found to inhibit the malonyl-CoA effect; however, the concentration of cyanide causing 50% inhibition was 1.25 mM; this was approximately 10 times greater than the concentration of cyanide causing 50% inhibition of the stearyl-CoA-enhanced rate. Lastly, the increase in the reoxidation rate by malonyl-CoA and ATP was not further enhanced by the addition of exogenous unsaturated and saturated fatty acids. The results are discussed in light of the two microsmal fatty acid biotransformation reactions, elongation and 9 desaturation.